Abstract
Serpins regulate coagulation and inflammation, binding serine proteases in suicide-inhibitory complexes. Target proteases cleave the serpin reactive center loop scissile P1-P1' bond, resulting in serpin-protease suicide-inhibitory complexes. This inhibition requires a near full-length serpin sequence. Myxomavirus Serp-1 inhibits thrombolytic and thrombotic proteases, whereas mammalian neuroserpin (NSP) inhibits only thrombolytic proteases. Both serpins markedly reduce arterial inflammation and plaque in rodent models after single dose infusion. In contrast, Serp-1 but not NSP improves survival in a lethal murine gammaherpesvirus68 (MHV68) infection in interferon γ-receptor-deficient mice (IFNγR(-/-)). Serp-1 has also been successfully tested in a Phase 2a clinical trial. We postulated that proteolytic cleavage of the reactive center loop produces active peptide derivatives with expanded function. Eight peptides encompassing predicted protease cleavage sites for Serp-1 and NSP were synthesized and tested for inhibitory function in vitro and in vivo. In engrafted aorta, selected peptides containing Arg or Arg-Asn, not Arg-Met, with a 0 or +1 charge, significantly reduced plaque. Conversely, S-6 a hydrophobic peptide of NSP, lacking Arg or Arg-Asn with -4 charge, induced early thrombosis and mortality. S-1 and S-6 also significantly reduced CD11b(+) monocyte counts in mouse splenocytes. S-1 peptide had increased efficacy in plasminogen activator inhibitor-1 serpin-deficient transplants. Plaque reduction correlated with mononuclear cell activation. In a separate study, Serp-1 peptide S-7 improved survival in the MHV68 vasculitis model, whereas an inverse S-7 peptide was inactive. Reactive center peptides derived from Serp-1 and NSP with suitable charge and hydrophobicity have the potential to extend immunomodulatory functions of serpins.
Highlights
Serpins are ubiquitous, complex, and highly active regulatory molecules that effectively control multiple coagulation, inflammatory, and neuroendocrine pathways [1,2,3]
This same reactive center loop (RCL) can insert into the neighboring -sheet A in other serpins in serpinopathies, causing serpin aggregates induced by genetic mutations and causing disease as for anti-thrombin III (SERPIN C1, ATIII), ␣-1 antitrypsin (SERPIN A1, AAT), and neuroserpin (SERPIN I1, NSP)
The serpin-derived RCL peptides did demonstrate some inhibition of plasminogen activator inhibitor-1 (PAI-1)
Summary
Angiotensinogen is cleaved first by renin and subsequently by the angiotensin-converting enzyme to form angiotensin I and angiotensin II, angiotensin II being a potent vasoconstrictor with a central role in regulation of blood pressure. When purified Serp-1 or NSP protein was given as a single bolus infusion immediately after aortic allograft transplant, both serpins significantly reduced plaque growth and inflammatory cell invasion at a 4-week follow-up [16]. The short plasma half-life of Serp-1 (20 min to 1 h) indicates probable rapid metabolism; beneficial effects are detectable weeks to months later in animal models, suggesting extended function. These extended functions may be produced by active metabolites. Inhibitory activity for RCL-derived Serp-1 and NSP peptides was assessed in vitro in human monocytes and in vivo in mouse ascites, aortic allograft transplant, and lethal mouse herpesviral (MHV68) vasculitis models
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